History of Modern Biotechnology I

207 Pages·2000·1.56 MB·English

by  A. Fiechter, T. Beppu, V.S. Bisaria, A.L. Demain, A. Fang, T.K. Ghose, J. Hollo, U.P. Kralovanszky, H. Kumagai, M. Roehr

Preface
The aim of the Advances of Biochemical Engineering/Biotechnology is to keep
the reader informed on the recent progress in the industrial application of
biology. Genetical engineering, metabolism ond bioprocess development includ-
ing analytics, automation and new software are the dominant fields of interest.
Thereby progress made in microbiology, plant and animal cell culture has been
reviewed for the last decade or so.
The Special Issue on the History of Biotechnology (splitted into Vol. 69 and 70)
is an exception to the otherwise forward oriented editorial policy. It covers a time
span of approximately fifty years and describes the changes from a time with
rather characteristic features of empirical strategies to highly developed and
specialized enterprises. Success of the present biotechnology still depends on
substantial investment in R & D undertaken by private and public investors,
researchers, and enterpreneurs. Also a number of new scientific and business
oriented organisations aim at the promotion of science and technology and the
transfer to active enterprises, capital raising, improvement of education and
fostering international relationships. Most of these activities related to modern
biotechnology did not exist immediately after the war. Scientists worked in
small groups and an established science policy didn’t exist.
This situation explains the long period of time from the detection of the anti-
biotic effect by Alexander Fleming in 1928 to the rat and mouse testing by Brian
Chain and Howart Florey (1940). The following developments up to the produc-
tion level were a real breakthrough not only biologically (penicillin was the first
antibiotic) but also technically (first scaled-up microbial mass culture under
sterile conditions). The antibiotic industry provided the processing strategies
for strain improvement (selection of mutants) and the search for new strains
(screening) as well as the technologies for the aseptic mass culture and down-
stream processing. The process can therefore be considered as one of the major
developments of that time what gradually evolved into “Biotechnology” in the
late 1960s. Reasons for the new name were the potential application of a “new”
(molecular) biology with its “new” (molecular) genetics, the invention of elec-
tronic computing and information science. A fascinating time for all who were
interested in modern Biotechnology.
True gene technology succeeded after the first gene transfer into Escherichia
coli in 1973. About one decade of hard work and massive investments were
necessary for reaching the market place with the first recombinant product.
Since then gene transfer in microbes, animal and plant cells has become a well-
X Preface
established biological technology. The number of registered drugs for example
may exceed some fifty by the year 2000.
During the last 25 years, several fundamental methods have been developed.
Gene transfer in higher plants or vertebrates and sequencing of genes and entire
genomes and even cloning of animals has become possible.
Some 15 microbes, including bakers yeast have been genetically identified.
Even very large genomes with billions of sequences such as the human genome
are being investigated. Thereby new methods of highest efficiency for sequenc-
ing, data processing, gene identification and interaction are available representing
the basis of genomics – together with proteomics, a new field of biotechnology.
However, the fast developments of genomics in particular did not have just
positive effects in society. Anger and fear began. A dwindling acceptance of
“Biotechnology” in medicine, agriculture, food and pharma production has
become a political matter. New legislation has asked for restrictions in genome
modifications of vertebrates, higher plants, production of genetically modified
food, patenting of transgenic animals or sequenced parts of genomes. Also
research has become hampered by strict rules on selection of programs,
organisms, methods, technologies and on biosafety indoors and outdoors.
As a consequence process development and production processes are of a high
standard which is maintained by extended computer applications for process
control and production management. GMP procedures are now standard and
prerequisites for the registation of pharmaceuticals. Biotechnology is a safe tech-
nology with a sound biological basis,a high-tech standard,and steadily improving
efficiency. The ethical and social problems arising in agriculture and medicine are
still controversial.
The authors of the Special Issue are scientists from the early days who are
familiar with the fascinating history of modern biotechnology.They have success-
fully contributed to the development of their particular area of specialization
and have laid down the sound basis of a fast expanding knowledge. They were
confronted with the new constellation of combining biology with engineering.
These fields emerged from different backgrounds and had to adapt to new
methods and styles of collaboration.
The historical aspects of the fundamental problems of biology and engineering
depict a fascinating story of stimulation, going astray, success, delay and satis-
faction.
I would like to acknowledge the proposal of the managing editor and the
publisher for planning this kind of publication. It is his hope that the material
presented may stimulate the new generations of scientists into continuing the re-
warding promises of biotechnology after the beginning of the new millenium.
Zürich, August 2000 Armin Fiechter
The Natural Functions of Secondary Metabolites
Arnold L. Demain, Aiqi Fang
Fermentation Microbiology Laboratory, Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA
E-mail:
2 A.L. Demain · A. Fang
3.8.2 Germination of Spores . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.8.3 Other Relationships Between Differentiation
and Secondary Metabolism . . . . . . . . . . . . . . . . . . . . . . . 32
3.9 Miscellaneous Functions . . . . . . . . . . . . . . . . . . . . . . . . 33
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
1
History of Secondary Metabolism
The practice of industrial microbiology (and biotechnology) has its roots deep
in antiquity [1]. Long before their discovery, microorganisms were exploited to
serve the needs and desires of humans, i.e., to preserve milk, fruit, and vege-
tables, and to enhance the quality of life with the resultant beverages, cheeses,
bread, pickled foods, and vinegar. In Sumeria and Babylonia, the oldest biotech-
nology know-how, the conversion of sugar to alcohol by yeasts,was used to make
beer. By 4000 BC, the Egyptians had discovered that carbon dioxide generated
by the action of brewer’s yeast could leaven bread, and by 100 BC, ancient Rome
had over 250 bakeries which were making leavened bread. Reference to wine,
another ancient product of fermentation, can be found in the Book of Genesis,
where it is noted that Noah consumed a bit too much of the beverage. Wine was
made in Assyria in 3500 BC As a method of preservation, milk was converted to
lactic acid to make yoghurt, and also into kefir and koumiss using Kluyveromyces
species in Asia. Ancient peoples made cheese with molds and bacteria. The use
of molds to saccharify rice in the Koji process dates back at least to 700 AD By
the 14th century AD, the distillation of alcoholic spirits from fermented grain, a
practice thought to have originated in China or The Middle East, was common
in many parts of the world. Interest in the mechanisms of these processes result-
ed in the later investigations by Louis Pasteur which not only advanced micro-
biology as a distinct discipline but also led to the development of vaccines and
concepts of hygiene which revolutionized the practice of medicine.
In the seventeenth century, the pioneering Dutch microscopist Antonie van
Leeuwenhoek, turning his simple lens to the examination of water, decaying
matter, and scrapings from his teeth, reported the presence of tiny “animal-
cules”, i.e., moving organisms less than one thousandth the size of a grain of
sand. Most scientists thought that such organisms arose spontaneously from
nonliving matter. Although the theory of spontaneous generation, which had
been postulated by Aristotle among others, was by then discredited with respect
to higher forms of life, it did seem to explain how a clear broth became cloudy via
growth of large numbers of such “spontaneously generated microorganisms”
as the broth aged. However, three independent investigators, Charles Cagniard
de la Tour of France, Theodor Schwann, and Friedrich Traugott Kützing of
Germany, proposed that the products of fermentation, chiefly ethanol and
carbon dioxide, were created by a microscopic form of life. This concept was
bitterly opposed by the leading chemists of the period (such as Jöns Jakob
Berzelius, Justus von Liebig, and Friedrich Wöhler), who believed fermentation
The Natural Functions of Secondary Metabolites 3
was strictly a chemical reaction; they maintained that the yeast in the fermenta-
tion broth was lifeless, decaying matter. Organic chemistry was flourishing at the
time, and these opponents of the living microbial origin were initially quite
successful in putting forth their views. It was not until the middle of the nine-
teenth century that Pasteur of France and John Tyndall of Britain demolished
the concept of spontaneous generation and proved that existing microbial life
comes from preexisting life. It took almost two decades, from 1857 to 1876, to
disprove the chemical hypothesis. Pasteur had been called on by the distillers of
Lille to find out why the contents of their fermentation vats were turning sour.
He noted through his microscope that the fermentation broth contained
not only yeast cells but also bacteria that could produce lactic acid. One of his
greatest contributions was to establish that each type of bioprocess is mediated
by a specific microorganism. Furthermore, in a study undertaken to determine
why French beer was inferior to German beer, he demonstrated the existence of
strictly anaerobic life, i.e., life in the absence of air.
The field of biochemistry originated in the discovery by the Buchners
that cell-free yeast extracts could convert sucrose into ethanol. Later, Chaim
Weizmann of the UK applied the butyric acid bacteria, used for centuries for
the retting of flax and hemp, for production of acetone and butanol. His use of
Clostridium during World War I to produce acetone and butanol was the first
nonfood bioproduct developed for large-scale production; with it came the
problems of viral and microbial contamination that had to be solved. Although
use of this process faded because it could not compete with chemical means
for solvent production, it did provide a base of experience for the development
of large scale cultivation of fungi for production of citric acid after the First
World War,an aerobic process in which Aspergillus niger was used. Not too many
years later, the discoveries of penicillin and streptomycin and their commercial
development heralded the start of the antibiotic era.
For thousands of years, moldy cheese, meat, and bread were employed in
folk medicine to heal wounds. It was not until the 1870s, however, that Tyndall,
Pasteur, and William Roberts, a British physician, directly observed the antago-
nistic effects of one microorganism on another. Pasteur, with his characteristic
foresight, suggested that the phenomenon might have some therapeutic poten-
tial. For the next 50 years, various microbial preparations were tried as medi-
cines, but they were either too toxic or inactive in live animals. The golden era
of antibiotics no doubt began with the discovery of penicillin by Alexander
Fleming [2] in 1929 who noted that the mold Penicillium notatum killed his
cultures of the bacterium Staphylococcus aureus when the mold accidentally
contaminated the culture dishes.After growing the mold in a liquid medium and
separating the fluid from the cells, he found that the cell-free liquid could inhibit
the bacteria. He gave the active ingredient in the liquid the name “penicillin”
but soon discontinued his work on the substance. The road to the development
of penicillin as a successful drug was not an easy one. For a decade, it remained
as a laboratory curiosity – an unstable curiosity at that. Attempts to isolate
penicillin were made in the 1930s by a number of British chemists, but the
instability of the substance frustrated their efforts. Eventually, a study began in
1939 at the Sir William Dunn School of Pathology of the University of Oxford by
4 A.L. Demain · A. Fang
Howard W. Florey, Ernst B. Chain, and their colleagues which led to the success-
ful preparation of a stable form of penicillin and the demonstration of its remark-
able antibacterial activity and lack of toxicity in mice. Production of penicillin
by the strain of Penicillium notatum in use was so slow, however, that it took over
a year to accumulate enough material for a clinical test on humans [3].When the
clinical tests were found to be successful, large-scale production became essen-
tial.Florey and his colleague Norman Heatley realized that conditions in wartime
Britain were not conducive to the development of an industrial process for
producing the antibiotic. They came to the US in the summer of 1941 to seek
assistance and convinced the US Department of Agriculture in Peoria, Illinois,
and several American pharmaceutical companies, to develop the production of
penicillin. Heatley remained for a period at the USDA laboratories in Peoria to
work with Moyer and Coghill.
Penicillin was originally produced in surface culture, but titers were very low.
Submerged culture soon became the method of choice. The use of corn-steep
liquor as an additive and lactose as carbon source stimulated production
further. Production by a related mold, Penicillium chrysogenum, soon became a
reality. Genetic selection began with Penicillium chrysogenum NRRL 1951, the
well-known isolate from a moldy cantaloupe obtained in a Peoria market. It was
indeed fortunate that the intense development of microbial genetics began in
the 1940s when the microbial production of penicillin became an international
necessity due to World War I. The early basic genetic studies concentrated
heavily on the production of mutants and the study of their properties. The ease
with which “permanent”characteristics of microorganisms could be changed by
mutation and the simplicity of the mutation technique had tremendous appeal to
microbiologists. Thus began the cooperative “strain-selection” program among
workers at the U.S. Department of Agriculture in Peoria, the Carnegie Institu-
tion, Stanford University, and the University of Wisconsin, followed by the
extensive individual programs that still exist today in industrial laboratories
throughout the world. By the use of strain improvement and medium modifica-
tions, the yield of penicillin was increased 100-fold in 2 years. The penicillin
improvement effort was the start of a long “engagement” between genetics and
industrial microbiology which ultimately proved that mutation is the major
factor involved in the hundred- to thousand-fold increases obtained in produc-
tion of microbial metabolites.
Strain NRRL 1951 of P. chrysogenum was capable of producing 60 µg/ml of
penicillin. Cultivation of spontaneous sector mutants and single-spore isola-
tions led to higher-producing cultures. One of these, NRRL 1951–1325, produc-
ed 150 mg/ml. It was next subjected to X-ray treatment by Demerec of the
Carnegie Institute at Cold Spring Harbor, New York, and mutant X-1612 was
obtained, which formed 300 mg/ml. This tremendous cooperative effort among
universities and industrial laboratories in England and the United States lasted
throughout the war. Further clinical successes were demonstrated in both
countries; finally in 1943 penicillin was used to treat those wounded in battle.
Workers at the University of Wisconsin isolated ultraviolet-induced mutants of
Demerec’s strain. One of these, Wis. Q-176, which produced 550 mg/ml, is the
parent of most of the strains used in industry today. The further development of
The Natural Functions of Secondary Metabolites 5
the “Wisconsin Family” of superior strains from Q-176 [4] led to strains produc-
ing over 1800 mg/ml. The new cultures isolated at the University of Wisconsin
and in the pharmaceutical industry did not produce the yellow pigment which
had been so troublesome in the early isolation of the antibiotic.
The importance of penicillin was that it was the first successful chemothera-
peutic agent produced by a microbe. The tremendous success attained in the
battle against disease with this compound not only led to the Nobel Prize being
awarded to Fleming, Florey, and Chain, but to a new field of antibiotics research,
and a new antibiotics industry. Penicillin opened the way for the development of
many other antibiotics, and yet it still remains the most active and one of the
least toxic of these compounds. Today, about 100 antibiotics are used to combat
infections to humans, animals, and plants.
The advent of penicillin, which signaled the beginning of the antibiotics era,
was closely followed by the discoveries of Selman A. Waksman, a soil micro-
biologist at Rutgers University. He and his students, especially H. Boyd Woodruff
and Hubert Lechevalier, succeeded in discovering a number of new antibiotics
from the the filamentous bacteria, the actinomycetes, such as actinomycin D,
neomycin and the best-known of these new “wonder drugs”, streptomycin.After
its discovery in 1944, streptomycin’s use was extended to the chemotherapy of
many Gram-negative bacteria and to Mycobacterium tuberculosis. Its major
impact on medicine was recognized by the award of the Nobel Prize to Waksman
in 1952. As the first commercially successful antibiotic produced by an actino-
mycete, it led the way to the recognition of these organisms as the most prolific
producers of antibiotics. Streptomycin also provided a valuable tool for study-
ing cell function. After a period of time, during which it was thought to act by
altering permeability, its interference with protein synthesis was recognized as
its primary effect. Its interaction with ribosomes provided much information on
their structure and function; it not only inhibits their action but also causes mis-
reading of the genetic code and is required for the function of ribosomes in
streptomycin-dependent mutants.
The development of penicillin fermentation in the 1940s marked the true
process beginning of what might be called the golden age of industrial micro-
biology, resulting in a large number of microbial primary and secondary
metabolites of commercial importance. Primary metabolism involves an inter-
related series of enzyme-mediated catabolic, amphibolic, and anabolic reactions
which provide biosynthetic intermediates and energy, and convert biosynthetic
precursors into essential macromolecules such as DNA, RNA, proteins, lipids,
and polysaccharides. It is finely balanced and intermediates are rarely accu-
mulated. The most important primary metabolites in the bio-industry are amino
acids, purine nucleotides, vitamins, and organic acids. Of all the traditional prod-
ucts made by bioprocess, the most important to human health are the secondary
metabolites (idiolites). These are metabolites which: (i) are often produced in a
developmental phase of batch culture (idiophase) subsequent to growth; (ii)
have no function in growth; (iii) are produced by narrow taxonomic groups of
organisms; (iv) have unusual and varied chemical structures; and (v) are often
formed as mixtures of closely related members of a chemical family. Bu’Lock [5]
interpreted secondary metabolism as a manifestation of differentiation which
6 A.L. Demain · A. Fang
accompanies unbalanced growth. In nature, their functions serve the survival
of the strain, but when the producing microorganisms are grown in pure
culture, the secondary metabolites have no such role. Thus, production ability in
industry is easily lost by mutation (“strain degeneration”). In general, both the
primary and the secondary metabolites of commercial interest have fairly low
molecular weights, i.e., less than 1500 daltons. Whereas primary metabolism is
basically the same for all living systems, secondary metabolism is mainly carried
out by plants and microorganisms and is usually strain-specific. The best-
known secondary metabolites are the antibiotics. More than 5000 antibiotics
have already been discovered, and new ones are still being found at a rate of
about 500 per year. Most are useless; they are either too toxic or inactive in living
organisms to be used. For some unknown reason, the actinomycetes are amaz-
ingly prolific in the number of antibiotics they can produce. Roughly 75% of all
antibiotics are obtained from these filamentous prokaryotes, and 75% of those
are in turn made by a single genus, Streptomyces.Filamentous fungi are also very
active in antibiotic production. Antibiotics have been used for purposes other
than human and animal chemotherapy, such as the promotion of growth of
farm animals and plants and the protection of plants against pathogenic micro-
organisms.
Cooperation on the development of the penicillin and streptomycin pro-
ductions into industrial processes at Merck & Co., Princeton University,
and Columbia University led to the birth of the field of biochemical engineer-
ing. Following on the heels of the antibiotic products was the development
of efficient microbial processes for the manufacture of vitamins (riboflavin,
cyanocobalamine, biotin), plant growth factors (gibberellins), enzymes (amylases,
proteases, pectinases), amino acids (glutamate, lysine, threonine, phenylalanine,
aspartic acid, tryptophan), flavor nucleotides (inosinate, guanylate), and poly-
saccharides (xanthan polymer), among others. In a few instances, processes have
been devised in which primary metabolites such as glutamic acid and citric acid
accumulate after growth in very large amounts. Cultural conditions are often
critical for their accumulation and in this sense, their accumulation resembles
that of secondary metabolites.
Despite the thousands of secondary metabolites made by microorganisms,
they are synthesized from only a few key precursors in pathways that comprise
a relatively small number of reactions and which branch off from primary
metabolism at a limited number of points. Acetyl-CoA and propionyl-CoA are
the most important precursors in secondary metabolism, leading to polyketides,
terpenes, steroids, and metabolites derived from fatty acids. Other secondary
metabolites are derived from intermediates of the shikimic acid pathway, the tri-
carboxylic acid cycle, and from amino acids. The regulation of the biosynthesis
of secondary metabolites is similar to that of the primary processes, involving
induction, feedback regulation, and catabolite repression [6].
There was a general lack of interest in the penicillins in the 1950s after the
exciting progress made during World War II. By that time, it was realized that
P. chrysogenum could use additional acyl compounds as side-chain precursors
(other than phenylacetic acid for penicillin G) and produce new penicillins,
but only one of these, penicillin V (phenoxymethylpenicillin), achieved any
The Natural Functions of Secondary Metabolites 7
commercial success. Its commercial application resulted from its stability to acid
which permitted oral administration, an advantage it held over the accepted
article of commerce, penicillin G (benzylpenicillin). Research in the penicillin
field in the 1950s was mainly of an academic nature, probing into the mechanism
of biosynthesis. During this period, the staphylococcal population was building
up resistance to penicillin via selection of penicillinase-producing strains and
new drugs were clearly needed to combat these resistant forms. Fortunately,
two developments occurred which led to a rebirth of interest in the penicillins
and related antibiotics. One was the discovery by Koichi Kato [7] of Japan in
1953 of the accumulation of the “penicillin nucleus” in P. chrysogenum broths
to which no side-chain precursor had been added. In 1959, Batchelor et al. [8]
isolated the material (6-aminopenicillanic acid) which was used to make “semi-
synthetic” (chemical modification of a natural product) penicillins with the
beneficial properties of resistance to penicillinase and to acid, plus broad-
spectrum antibacterial activity. The second development was the discovery of
“synnematin B” in broths of Cephalosporium salmosynnematum by Gottshall et
al. [9] in Michigan, and that of “cephalosporin N” from Cephalosporium sp. by
Brotzu in Sardinia and its isolation by Crawford et al. [10] at Oxford. It was soon
found that these two molecules were identical and represented a true penicillin
possessing a side-chain of d-a-aminoadipic acid. Thus, the name of this anti-
biotic was changed to penicillin N. Later, it was shown that a second antibiotic,
cephalosporin C, was produced by the same Cephalosporium strain producing
penicillin N [11].Abraham, Newton, and coworkers found the new compound to
be related to penicillin N in that it consisted of a b-lactam ring attached to a side
chain of d-a-aminoadipic acid. It differed, however, from the penicillins in con-
taining a six-membered dihydrothiazine ring in place of the five-membered
thiazolidine ring of the penicillins.
Although cephalosporin C contained the b-lactam structure, which is the
site of penicillinase action, it was a poor substrate and was essentially not
attacked by the enzyme, was less toxic to mice than penicillin G, and its mode
of action was the same; i.e., inhibition of cell wall formation. Its disadvantage
lied in its weak activity; it had only 0.1% of the activity of penicillin G against
sensitive staphylococci, although its activity against Gram-negative bacteria
equaled that of penicillin G. However, by chemical removal of its d-a-amino-
adipidic acid side chain and replacement with phenylacetic acid, a penicillinase-
resistant semisynthetic compound was obtained which was 100 times as active
as cephalosporin C. Many other new cephalosporins with wide antibacterial
spectra were developed in the ensuing years, making the semisynthetic cephalo-
sporins the most important group of antibiotics. The stability of the cephalos-
porins to penicillinase is evidently a function of the dihydrothiazine ring since:
(i) the d-a-aminoadipic acid side chain does not render penicillin N immune to
attack; and (ii) removal of the acetoxy group from cephalosporin C does not
decrease its stability to penicillinase. Cephalosporin C competitively inhibits
the action of penicillinase from Bacillus cereus on penicillin G. Although it does
not have a similar effect on the Staphylococcus aureus enzyme, certain of its
derivatives do. Cephalosporins can be given to some patients who are sensitive
to penicillins.